Screening Method for Dnak Inhibitors

Abstract

Screening methods for identification of inhibitors of DnaK activity are provided. Such inhibitors have utility as antibacterial agents.

1 . A screening method for identification of a DnaK inhibitor, comprising the steps of:
 (a) providing a denatured substrate protein in solution;   (b) contacting said substrate protein with DnaK, or a homolog thereof, in the presence and absence of a compound suspecting of having DnaK inhibitory activity; and   (c) determining whether the activity of said substrate protein in the presence of said compound is decreased relative to the activity of said substrate protein in the absence of said compound,   whereby a decrease in substrate protein activity indicates that said compound is a DnaK inhibitor.   
     
     
         2 . The screening method of  claim 1 , wherein the substrate protein is firefly luciferase. 
     
     
         3 . The screening method of  claim 2 , wherein the firefly luciferase is denatured in guanidinium hydrochloride. 
     
     
         4 . The screening method of  claim 2 , wherein the DnaK, or a homolog thereof, is derived from a bacterial genus selected from the group consisting of  Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, Bordetella  and  Francisella.    
     
     
         5 . The screening method of  claim 1 , wherein step (b) further includes DnaJ and GrpE. 
     
     
         6 . The screening method of  claim 5 , wherein the concentration of GrpE is greater than that of DnaK and DnaJ. 
     
     
         7 . The screening method of  claim 6 , wherein the concentration of GrpE is at least about 2-fold greater than that of DnaK and DnaJ. 
     
     
         8 . The screening method of  claim 7 , wherein the concentration of GrpE is about 2-fold greater than that of DnaK. 
     
     
         9 . The screening method of  claim 7 , wherein the concentration of GrpE is about 8-fold greater than that of DnaJ. 
     
     
         10 . The screening method of  claim 7 , wherein the concentration of GrpE is about 2-fold greater than that of DnaK and about 8-fold greater than that of DnaJ. 
     
     
         12 . The screening method of  claim 1 , wherein the compound is a peptide. 
     
     
         13 . The screening method of  claim 1 , wherein the compound is a derivative or analog of pyrrhocoricin. 
     
     
         14 . The screening method of  claim 1 , wherein the compound is a small organic compound. 
     
     
         15 . The screening method of  claim 14 , wherein the small organic compound is dissolved in an organic solvent prior to contacting the substrate protein with DnaK. 
     
     
         16 . The screening method of  claim 1 , further comprising confirming the antibacterial activity of the compound in a bacterial cell-based assay. 
     
     
         17 . The screening method of  claim 16 , wherein the confirmatory assay includes a bacterial strain having a partially compromised cell wall/membrane. 
     
     
         18 . The screening method of  claim 1 , further comprising a positive control for DnaK inhibition. 
     
     
         19 . The screening method of  claim 18 , wherein the positive control is a known peptide inhibitor. 
     
     
         20 . The screening method of  claim 19 , wherein the known peptide inhibitor is pyrrhocoricin or a derivative or an analog thereof. 
     
     
         21 . The screening method of  claim 20 , wherein the analog is CHP-105. 
     
     
         22 . The screening method of  claim 1 , further comprising a negative control for spontaneous refolding.